Ribosome biogenesis is an essential and complex multistep pathway which exists in all living cells. The precursor rRNA (pre-rRNA) encodes three structural RNAs that must be correctly modified, folded, processed and assembled with approximately 80 ribosomal proteins and acted upon by probably several hundred trans action factors during the assembly process. This intricate pathway yields the two mature ribosomal subunits comprising the functional ribosome. Severe mutations in this pathway are lethal. Minor perturbations are characterized by diseases including dyskeratosis congenital and some autoimmune diseases. The focus of the research in my lab is to learn more about the ribosomal assembly process by identifying and learning the in vivo functional roles of the cis sequences and trans factors required for processing within ITS2 in eukaryotes. Over the past year my lab has made progress on three fronts. First, we are using the genetics available in yeast, S.cerevisiae, to differentiate between two predicted structural models for an intramolecular interaction in pre-rRNA necessary for subsequent processing steps. Identification of the structural conformation and sequence recognition of primary cleavage sites within the precursor is essential for understanding how the structure affects ribosome biogenesis. Second, we are using biochemical and biophysical methods to identify and characterize proteins that comprise the Xenopus U8 small nucleolar ribonucleoprotein particle (U8 snoRNP), an essential trans-acting factor required for accumulation of newly formed large ribosomal subunits. Third, we are examining the kinetics and snoRNA:pre-rRNA interactions in pre-rRNA processing and directly testing our working model for the role of U8 RNA in vivo (1). One focus of the lab takes advantage of the genetics available in yeast, S.cerevisiae, to use our in vivo functional genetic assay to learn whether subtle sequence and structural mutations in pre-rRNA affects the efficiency or accuracy of pre-rRNA processing. Our early experiments in yeast unequivocally demonstrated that formation of a particular intramolecular interaction is critical for pre-rRNA processing (2, 3). We have since identified interconverting structural conformations of ITS2 which play critical roles in the maturation event (4). We are now generating constructs which will address requirements for structural conformation and sequence recognition at the primary cleavage site within ITS2. The information about cleavage site recognition will be combined with our knowledge about alternative structures of this region of the pre-rRNA to allow us to begin to order the timing of the structural alterations and assembly of trans-factors (including the processing machinery) to affect the efficiency of pre-rRNA processing. A second focus is a continuation of our characterization of trans-acting factors essential for pre-rRNA processing in vertebrates. U8 snoRNP is essential for pre-rRNA processing in Xenopus oocytes. In the absence of U8 RNA, pre-rRNA processing is inhibited and no mature rRNA accumulates (1). Mutagenesis of U8 RNA indicated that sequences at the 5 prime end of U8 RNA were necessary, but not sufficient to direct pre-rRNA processing; U8 RNP proteins affected the stability of the U8 RNA and the efficiency of processing (1). To better understand how the U8 RNP functions in vivo, we have been identifying proteins which specifically bind U8 RNA in vitro (5, 6). We recently reported our identification of a 29 kDa protein from Xenopus ovary extracts which specifically binds U8 RNA (5). We are continuing to characterize the X29 protein on biochemical, biophysical and cellular levels. We have recently demonstrated X29 is the first example of a nuclear decapping protein identified in eukaryotes. We have begun to look at this protein using structural methods (X-ray crystallography) to better understand how this protein binds U8 RNA. We are looking at the in vivo localization of this protein to better correlate the in vivo role with its identified in vitro function. Together the structural biology, biochemistry and cell biology will provide us with a better understanding of how this protein affects U8 snoRNA and thus ribosome biogenesis. A third focus of the lab has been a direct examination of the working model for U8 function in vivo. We previously proposed that U8 functioned in vivo as an RNA chaperone by facilitating an interaction between 5.8S and 28S in pre-rRNA, which persists in the mature ribosome (1). We have been generating constructs which will be used in vivo in Xenopus oocytes to directly assay the requirement for this proposed U8 base pairing. These experiments are particularly critical since the Xenopus oocyte is the only vertebrate model system for pre-rRNA processing. Using our two model systems and taking advantage of their differences will allow us to better understand the basic mechanisms of pre-rRNA assembly and the timing of the processing events. With an understanding of the normal processes and components involved in ribosome biogenesis we can begin to address what aspects are abnormal in diseases like dyskeratosis congenita, scleroderma and other cell-proliferation diseases where the rate of ribosome biogenesis has been altered. Identification of conserved and unique cis- and trans-acting components involved in ribosome biogenesis will provide additional components to monitor in diseases implicating defects in the complex process of ribosome biogenesis.